Conjugates of urolithin A with NSAIDs, their stability, cytotoxicity, and anti-inflammatory potential

Urolithin A (UA, 1), a gut microbiota postbiotic metabolite is attributed to express interesting biological activities indicated by in vitro, in vivo and clinical studies. Due to its strong anti-inflammatory properties it is considered as a promising lead molecule for further drug development, however, its strong phase II metabolism, severely limits its oral application. Therefore, monoesterified UA derivatives with selected NSAIDs: ibuprofen (Mix 3a/3b), mefenamic acid (Mix 4a/4b), diclofenac (Mix 5a/5b) and aspirin (Mix 6a/6b) were designed. Performed array of stability assays indicated Mix 4a/4b as a most suitable candidate for further studies due to its exceptional stability in human plasma. Thus, we evaluated effects of Mix 4a/4b on cell viability as well as the impact on cytokines secretion in THP-1 derived macrophages and compared it to UA. At high concentration (50 µM) Mix 4a/4b expressed a cytotoxic effect, however at concentration of 5 µM it significantly suppressed TNF-α secretion, and significantly increased ani-inflammatory IL-10 secretion at 10 µM without affecting cell viability. This work has led to selection of a novel UA derivatives, which are stable in solutions and in human plasma as well as posess anti-inflammatory activity towards THP-1 macrophages at non-cytotoxic concentrations.


Stability assay performance
Control experiments. UADs dissolved in DMSO were added to deionised water, mixed thoroughly and, immediately, samples were taken, added to 0.2% formic acid in acetonitrile and subjected to UHPLC-DAD-MS analysis. In experiments evaluating stability in human plasma, UADs dissolved in DMSO were added to plasma, mixed thoroughly and, immediately, samples were taken, added to 0.2% formic acid in acetonitrile, centrifuged at 13000 RPM for 10 minutes and supernatants were subjected to UHPLC-DAD-MS analysis.
Acid hydrolysis. UADs dissolved in DMSO were added to HCl (1M), mixed thoroughly and kept at 37 °C for 24h or 80 °C for 12h. Samples were taken, added to NaOH (1M), mixed with 0.2% formic acid in acetonitrile and subjected to UHPLC-DAD-MS analysis.
Base hydrolysis. UADs dissolved in DMSO were added to NaOH (1M), mixed thoroughly and kept at 37 °C for 24h or 80 °C for 12h. Samples were taken, added to HCl (1M), mixed with 0.2% formic acid in acetonitrile and subjected to UHPLC-DAD-MS analysis.
Thermal degradation. UADs dissolved in DMSO were added to deionised water, mixed thoroughly and kept at room temperature for 24h, 37 °C for 24h, 80 °C for 12h or -70 °C for 24h. Additionally, samples were exposed to a cycle consisting of incubation at -70 °C for 24h followed by 2h thawing in tap water at room temperature, subsequent freezing at -20 °C for 24h and 2h thawing in tap water at room temperature. Samples were taken, mixed with 0.2% formic acid in acetonitrile and subjected to UHPLC-DAD-MS analysis.
UV degradation. ADs dissolved in DMSO were added to deionised water, mixed thoroughly and kept at room temperature for 7h in plastic falcons and exposed to UV-C light (λ=254 nm, Osram G3058/OF lamp, Osram Licht AG, Munich, Germany). Samples were taken, mixed with 0.2% formic acid in acetonitrile and subjected to UHPLC-DAD-MS analysis.
Oxidative degradation. UADs dissolved in DMSO were added to 30% H2O2 and kept at 37 °C for 24h or 80 °C for 12h. Samples were taken, mixed with 0.2% formic acid in acetonitrile and subjected to UHPLC-DAD-MS analysis.

Stability in human plasma.
Human plasma was collected from three independent donors and obtained from the Warsaw Blood Donation Center. UADs dissolved in DMSO were added to plasma, mixed thoroughly and kept at 37 °C for 30, 90 and 240 minutes. Samples were taken, added to 0.2% formic acid in acetonitrile, centrifuged at 13000 RPM for 10 minutes and supernatants were subjected to UHPLC-DAD-MS analysis.  Table S1. Post hoc analysis of effects of UA and Mix 4a/4b on THP-1 derived macrophage viability measured using MTT assay 3 hours after stimulation. Cells viability presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -nonstimulated control, ST -LPS stimulated control Table S2. Post hoc analysis of effects of UA and Mix 4a/4b on THP-1 derived macrophage viability measured using MTT assay 24 hours after stimulation. Cells viability presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -non-stimulated control, ST -LPS stimulated control 9 Table S3. Post hoc analysis of effects of UA and Mix 4a/4b on THP-1 derived macrophage viability measured using NRU assay 3 hours after stimulation. Cells viability presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -non-stimulated control, ST -LPS stimulated control Table S4. Post hoc analysis of effects of UA and Mix 4a/4b on THP-1 derived macrophage viability measured using NRU assay 24 hours after stimulation. Cells viability presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -non-stimulated control, ST -LPS stimulated control Table S5. Post hoc analysis of effects of UA and Mix 4a/4b on THP-monocyte viability measured using PI flow cytometric assay after 24 hours incubation. ST group. NST -non-stimulated control, ST -LPS stimulated control Table S6. Post hoc analysis of effects of UA and Mix 4a/4b on TNF-α secretion in THP-1 derived macrophages 3 hours after stimulation. TNF-α secretion presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -non-stimulated control, ST -LPS stimulated control Table S7. Post hoc analysis of effects of UA and Mix 4a/4b on Il-10 secretion in THP-1 derived macrophages 24 hours after stimulation. TNF-α secretion presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -non-stimulated control, ST -LPS stimulated control Table S8. Post hoc analysis of effects of UA and Mix 4a/4b on Il-6 secretion in THP-1 derived macrophages 24 hours after stimulation. TNF-α secretion presented as a ratio of the experimental group to LPS stimulated cells. ST group. NST -non-stimulated control, ST -LPS stimulated control